Therapeutic use of agonist ligands specific to G2A receptor

ABSTRACT

The present invention relates to novel therapeutical use of agonist ligands specific to G2A receptor. More particularly, the present invention relates to methods for treating a disease or disorder associated with neutrophil accumulation and hyperactivity and/or excessive release of IL-8, or with microbial infection, in a subject, comprising administering LPC (lysophosphatidylcholine), SPC(sphingophosphorylcholine) or derivatives thereof to the subject. The agonist ligands for G2A receptor according to the present invention and pharmaceutical- or therapeutical composition comprising said ligands can be used effectively in treatment of a disease or disorder associated with neutrophil accumulation and hyperactivity and/or excessive release of IL-8, specifically inflammatory diseases and diseases associated with ischemia-reperfusion injury as well as microbial infection.

FIELD OF THE INVENTION

The present invention relates to novel therapeutic use of agonistligands specific to G2A receptor and more particularly, to methods fortreating a disease or disorder associated with neutrophil accumulationand -hyperactivity and/or excessive release of IL-8, or an infectiousdisease in a subject, comprising administering LPC(lysophosphatidylcholine), SPC (sphingophosphorylcholine) or derivativesthereof into the subject.

BACKGROUND OF THE INVENTION

Inflammation is an important defense response occurring in body againstpathogens, foreign substances and tissue injury. Inflammationaccompanies systemic symptoms such as fever, weakness, loss of appetiteand chill, or local symptoms such as redness, swelling, pain anddysfunction. Inflammation is divided into acute inflammation, subacuteinflammation and chronic inflammation according to its duration. Acuteinflammation reaction occurs in blood vessel and is mostly mediated byneutrophils. Especially, in the case of suppurative inflammation,remarkable increase of neutrophils is observed. Chronic inflammation iscontinued for several weeks or several months. It is different fromacute inflammation in that injury and recovery of tissues occur at thesame time (Robbins Pathological Basis of Disease by R. S. Cotran, VKumar, and S. L. Robbins, W.B. Saunders Co., p. 75, 1989). Althoughchronic inflammation may be derived directly from acute inflammation, itgenerally results from continuous infections that cause prolongedhypersensitive reactions (ex., tuberculosis, syphilis, fungalinfection), exposure to continuous endotoxin (ex., increased plasmalipids) or exotoxins (ex., silica, asbestos, tar, surgery sutures), orautoimmune response against self-tissues (ex., rheumatic arthritis,systemic lupus erythematosus, multiple sclerosis, psoriasis) and it canthus initiate insidious onset that proceeds as times goes by.Accordingly, chronic inflammation includes numerous medical symptomssuch as rheumatic arthritis, restenosis, psoriasis, multicentricsclerosis, surgery synechia, tuberculosis and chronic inflammatory lungdiseases (ex., asthma, pneumoconiosis, chronic occlusive lung disease,pulmonary fibrosis). Subacute inflammation refers to an inflammationbetween acute and chronic inflammations.

As main inflammatory diseases, there are rhinitis and sinusitis such asinfectious rhinitis, allergic rhinitis, chronic rhinitis, acutesinusitis and chronic sinusitis; otitis media such as acute purulentotitis media and chronic purulent otitis media; pneumonia such asbacterial pneumonia, bronchopneumonia, lobar pneumonia, Legionellapneumonia and viral pneumonia; acute or chronic gastritis; enteritissuch as infectious enterocolitis, Crohn's disease, idiopathic ulcerativecolitis and pseudomembranous colitis; and arthritis such as pyogenicarthritis, tuberculous arthritis, degenerative arthritis and rheumatoidarthritis. In addition, there is sepsis that accompanies extremesystemic inflammatory reaction at early stage. This sepsis results fromexcessive reaction of hosts against endotoxin of gram-negative bacteria,etc. In prior arts, so as to treat sepsis, there have been usedantibiotics and steroid preparations, but their effects are weak andthus the death rate of hosts due to septicemia is still high.

Also, excessive inflammation causes permanent injury of surroundingtissues, and acute respiratory distress syndrome (ARDS) is regarded asone of typical inflammatory diseases resulting from tissue injury byexcessive inflammation. The ARDS is an acute hypoxemic respiratoryfailure due to pulmonary edema resulting from the increased permeabilityof alveolar capillary barrier. It is regarded as the most severe case inacute lung injury (ALI). Clinical symptoms that lead patients to therisk of ARDS are various, for example, trauma, bleedings or septicemia,and ARDS results from excessive systemic inflammation reaction due tothese symptoms. Even though there have been conducted treatments such astreatment of hypoxia, endotracheal intubation, mechanical ventilation,etc., the death rate due to ARDS still reaches 50˜70%. Circulatinginflammatory cells, especially neutrophils, have been known to have animportant role in initiation and development of acute lung injury, e.g.,pulmonary edema, inflammation reaction, etc. (Abraham et al., Am. J.Physiol., 279, L1137-L1145, 2000). Several scientists proved thatneutrophils are extensively accumulated in lungs of ARDS patients(Weinacker & Vaszar, Annu. Rev Med., 52: 221-37, 2001). Theseneutrophils, once activated, discharge proteases including matrixmetalloproteinases and other mediators causing lung injury. Hence, ifneutrophil accumulation in lung is suppressed, ARDS due to acute lunginjury may be treated.

Multiple organ dysfunction syndrome (MODS) is a disease resulting fromthe complication of sepsis, etc. As examples of MODS, there can beincluded acute hepatic failure, acute renal failure, lung failure,gastrointestinal bleeding, etc. For the treatment of MODS, antibioticsand steroid preparations have been used, however, their effects areweak.

Meanwhile, neutrophils (also called ‘polymorphonuclear leucocytes(PMNs)’) are phagocytic cells that have an important role in hostdefense mechanism and occupy approximately 60% of leucocytes that arecirculating in body. The membrane of neutrophils has receptors forhemopoietic growth factors such as GM-CSF (granulocyte macrophage-colonystimulating factor), G-CSF (granulocyte-colony stimulating factor),etc., G proteins involving in signal transduction associated withreceptors for opsonin and chemotactic factors, ion channels associatedwith the ion exchange of Na⁺, K⁺, Ca²⁺, etc., enzymes and phospholipids.Also, at the surface of neutrophils, there exist Fc receptors against anIgG antibody such as CD16 and CD32 and receptors to C3 complementproteins such as CR1 and CR3. Accordingly, antigens bound thereto can beeasily recognized and eliminated. As within the granule of neutrophils,there are defensin associated with disinfection such as peroxidase,lactoferrin, leukocyte adhesion receptor and alkaline phosphatase, andbactericidal/permeability-increasing protein (BPI), which destroyinfectious agents or are involved in the proceedings of inflammatoryreaction. Besides, the neutrophils express cell adhension proteins suchas CD11a/CD18(LFA-1), selectin, etc., which have an important role inthe movement of neutrophils in inflammatory reaction.

For a normal adult, neutrophils are produced in an amount of0.85-1.6×10⁹ cells/kg/day. After being produced and differentiated inbone marrow over approximately 14 days, they enter peripheral bloods andcirculate there for about 6 hours. They penetrate then into tissues anddie or are lost at mucous membranes after surviving for several days inthe tissues. Neutrophils have a short half-life of about 6-10 hours andthey are removed in macrophages by apoptosis. The neutrophil apoptosisoccurs spontaneously or by the external stimulus. As a typical exampleof the external stimulus, there can be mentioned a Fas pathway. Fas is asubstance similar to TNF receptor that exists at the surface ofneutrophils, and it induces apoptosis via an FADD pathway in cells onceit is stimulated by a Fas ligand. Caspase has been known to have animportant role in such a pathway. Neutrophil apoptosis has been known tobe delayed or suppressed by various inflammation mediators. Some reportsproposed that the delay (suppression) of neutrophil apoptosis observedin ARDS is due to GM-CSF. However, the intracellular transductionpathway that delays apoptosis of neutrophil had been hardly known. Thatis, in several inflammatory diseases, various inflammation mediatorssuppress neutrophil apoptosis that is physiologically and activelyoccurring, and consequently, continuous inflammatory reaction occurs byexcessive neutrophil accumulation resulting in the damage to surroundingtissues. As inflammation mediators, there have been known G-CSF, GM-CSF,IFN-γ, IL-2, IL-6, etc. that are endogenous factors as well as LPS(lipopolysaccharide) that is derived from outside of the body.

Recently, numerous studies about LPC (lysophosphatidylcholine) and SPC(sphingosylphosphorylcholine) having LPA (lysophosphatidyl acid) or S1P(sphingosine 1-phosphate), which have been known as lipid transmitter,and a choline bound thereto, are being under progress. LPC and SPC havebeen known to have an important role in functioning not only asintermediates in biosynthesis of cellular membranes, together with LPAand SIP, but also as signaling molecules (Fukushima, N. et al., Annu.Rev. Pharmacol. Toxicol., 41: 507-534, 2001). They are bound to theirreceptors and induce various cell reactions such as cell proliferation,differentiation, movement, cell death, etc. through several signaltransductions (Lynch, K. R. et al., Trends Pharmacol. Sci., 20(12):473-475, 1999). The receptors to which they are bound are a kind ofreceptors that are classified as G protein-coupled receptors. SinceOGR-1 (orphan G-protein-coupled receptor 1) was for the first time foundas a receptor of SPC (Xu, Y. et al., Nat. Cell. Biol., 2(5):264-267,2000), studies about identifying ligands for GPR4- and G2A receptorshaving a structure similar to OGR-1 have been conducted. As a result, itwas identified that GPR4 recognized SPC and LPC as its ligand and GPR4promoted cell proliferation by SPC and LPC whereas OGR-1 suppressed cellproliferation by SPC (Zhu, K. et al., J. Biol. Chem., 276(44):41325-41335, 2001). Moreover, it was reported that G2A had a highaffinity to LPC, however, it had a low affinity to SPC (Kabarowski, J.H. et al., Science, 293(5530): 702-705, 2001). G2A is mostly found inlymphocytes and the expression thereof is upregulated by stress andprolonged mitogenic signals. It was reported that in knockout mice thatdid not have G2A receptors, autoimmune diseases were caused (Le, L. Q.et al., Immunity, 14(5): 561-571, 2001).

Hence, in the course of conducting continuous studies to find out newtherapeutic agents for treating inflammatory diseases, the presentinventors identified that agonist ligands specific to G2A receptor thatexists in neutrophils block suppression of neutrophil apoptosis byinflammation mediators and release of IL-8 (interleukin-8) inneutrophils and monocytes, and exhibit excellent therapeutic effect onan inflammatory disease, especially inflammatory diseases associatedwith hyperactivity of neutrophil and excessive release of IL-8, or adisease associated with microbial infection, and thus they havecompleted the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for inducing neutrophil apoptosis in cells, tissues or a bodyusing an agonist ligand specific to G2A receptor.

It is another object of the invention to provide a method for inhibitingthe excessive release of IL-8 in cells, tissues or a body using anagonist ligand specific to G2A receptor.

It is further object of the invention to provide a method for increasingthe bactericidal activity of neutrophils using an agonist ligandspecific to G2A receptor.

It is still further object of the invention to provide a method fortreating a disease or disorder associated with suppression of neutrophilapoptosis or excessive release of IL-8 using an agonist ligand specificto G2A receptor.

Further, it is another object of the invention to provide apharmaceutical or therapeutical composition comprising an agonist ligandspecific to G2A receptor as an active ingredient.

It is still another object of the invention to provide a noveltherapeutic use of an agonist ligand specific to G2A receptor.

To achieve the object above, the present invention provides a method forinducing neutrophil apoptosis in cells, tissues or a body, comprisingadministering an agonist ligand specific to G2A receptor into the cells,tissues or body in an amount effective to induce neutrophil apoptosis.

To achieve another object of the invention, the invention provides amethod for inhibiting release of IL-8 in cells, tissues or a body,comprising administering an agonist ligand specific to G2A receptor intothe cells, tissues or body in an amount effective to inhibit release ofIL-8.

To achieve another object of the invention, the invention provides amethod for increasing bactericidal activity of neutrophils in cells,tissues or a body, comprising administering an agonist ligand specificto G2A receptor into the cells, tissues or body in an amount effectiveto increase bactericidal activity of neutrophils.

To achieve another object of the invention, the invention provides amethod for treating or preventing a disease or disorder associated withsuppression of neutrophil apoptosis or excessive release of IL-8 in asubject, comprising administering an agonist ligand specific to G2Areceptor into the subject.

To achieve another object of the invention, the invention provides apharmaceutical composition for inducing neutrophil apoptosis orinhibiting release of IL-8 comprising an agonist ligand specific to G2Areceptor as an active ingredient.

To achieve another object of the invention, the invention provides acomposition for treating or preventing a disease or disorder associatedwith suppression of neutrophil apoptosis or excessive release of IL-8comprising an agonist ligand specific to G2A receptor as an activeingredient.

Further, to achieve another object of the invention, the inventionprovides a use of an agonist ligand specific to G2A receptor for themanufacture of a pharmaceutical composition for inducing neutrophilapoptosis or inhibiting release of IL-8 in cells, tissues or a body.

To achieve another object of the invention, the invention provides a useof an agonist ligand specific to G2A receptor for the manufacture of anagent for treating a disease or disorder associated with the suppressionneutrophil apoptosis or excessive release of IL-8.

The present invention will be described in detail.

The agonist ligands specific to G2A receptor according to the presentinvention include LPC (lysophosphatidylcholine), SPC(sphingosylphosphorylcoline) and derivatives thereof.

The LPC as used herein is represented by the following formula I:

wherein R₁ is an alkyl Of C₄₋₃₀ or an alkenyl Of C₄₋₃₀ having one ormore double bonds. Preferably, the LPC is not limited to, but may beselected from the group consisting of 1-stearoyl (18:0)lysophosphatidylcholine (_(L-α)-Lysophosphatidylcholine stearoyl;Lysolecithin stearoyl), 1-oleoyl (18:1) lysophosphatidylcholine(_(L-α)-Lysophosphatidylcholine oleoyl; Lysolecithin oleoyl),1-myristoyl (14:0) lysophospatidylcholine(_(L-α)-Lysophosphatidylcholine myristoyl), and 1-palmitoyl (16:0)lysophosphatidylcholine (_(L-α)-Lysophosphatidylcholine palmitoyl;Lysolecithin palmitoyl; _(DL-α)-Lysophosphatidylcholine palmitoyl).

Also, the SPC as used herein is represented by the following formula II:

In the SPC of the above formula II, the number of terminal carbons inthe sphingosine portion may be from 4 to 30.

Further, in the present invention, the derivatives of LPC or SPC may beused. Preferably, they may be ether derivatives of LPC represented bythe following formula III:

wherein R₂ is an alkyl of C₄₋₃₀ or an alkenyl of C₄₋₃₀ having one ormore double bonds. More preferably, they are not limited to, but may beselected from the group consisting of_(L-α)-lysophosphatidylcholine-_(γ)-O-alk-1-enyl(Lysophosphatidalcholine), _(L-α)-lysophosphatidylcholine-_(γ)-O-alkyl(Lyso-platelet activating factor),_(DL-α)-lysophosphatidylcholine-_(γ)-O-hexadecyl (rac-Lyso-plateletactivating factor), and _(L-α)-lysophosphatidylcholine-_(γ)-O-hexadecyl(Lyso-platelet activating factor; Lyso-PAF-C₁₆).

The LPC, SPC and derivatives thereof are commercially available withease. Specifically, they can be purchased from Sigma Chemical Co. (USA).Further, they may be isolated from animals and also can be preparedaccording to synthetic methods well known in the pertinent art. The LPC,SPC and derivatives thereof are endogenous substances in mammal, andthus their safety is as good as proven.

The agonist ligands specific to G2A receptor according to the presentinvention largely show two activities. First, they do not show in vitrodirect antibacterial effect but have in vivo functions of blocking bothsuppressed apoptosis of neutrophils and release of IL-8 in neutrophilsand monocytes. Therefore, they can be effectively used in treatment orprevention of a disease or disorder associated with suppression ofneutrophil apoptosis and/or excessive release of IL-8.

Second, the agonist ligands specific to G2A receptor according to thepresent invention enable neutrophils to kill pathogens more readily byincreasing their bactericidal ability. In this respect, the agonistligands according to the invention can be said as a new type ofantibacterial agent because they do not show direct killing effectsagainst pathogens but enable neutrophils to eliminate pathogens morereadily. Therefore, the agonist ligands according to the invention canbe used as a therapeutic agent or therapeutic additive for variousdiseases associated with microbial infection.

The pharmaceutical composition, or therapeutic or preventive compositioncomprising an agonist ligand specific to G2A receptor according to theinvention may further comprise pharmaceutically acceptable carriers, forexample, carriers for oral administration or for parenteraladministration. The carriers for oral administration may includelactose, starch, cellulose derivatives, magnesium stearate, stearic acidand so on. For oral administration, the agonist ligands specific to G2Areceptor according to the invention can be used in the form ofintake-type tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, etc. in an admixture of excipients. Also,the carriers for parenteral administration may comprise water, suitableoils, salines, water-soluble glucose and glycols, etc, and may furthercomprise stabilizer or preserver. As a suitable stabilizer, there areantioxidants such as ascorbic acid, sodium sulfite or sodium hydrogensulfite. As a suitable preserver, there are benzalkonium chloride,methyl- or propyl-paraben and chlorobutanol. For other pharmaceuticallyacceptable carriers, the following literature can be consulted:Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company,Easton, Pa., 1995.

The pharmaceutical composition or therapeutical composition according tothe present invention can be formulated into various parenteral or oraldosage forms. Typical dosage form for parenteral administration is adosage form for injection, preferably, an isotonic aqueous solution or asuspension. The dosage form for injection can be prepared using suitabledispersion agent, wetting agent or suspension agent according to theknown methods in the pertinent art. For example, each ingredient isdissolved in saline or buffer, and then can be formulated into a dosageform for injection. Also, typical dosage form for oral administration istablets, capsules, etc., which may comprise diluents (Ex.: lactose,dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), orlubricants (Ex.: silica, talc, stearic acid and magnesium or calciumsalt thereof and/or polyethylene glycol) in addition to the activeingredient. Furthermore, the tablets may further comprise binders suchas magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidine. If necessary, they may further comprisedisintegrants or effervescent mixtures such as starch, agar, alginicacid or sodium salt thereof, and/or absorbents, colorants, flavors andsweeteners. Such dosage form can be prepared by conventional mixing,granulation or coating methods.

The pharmaceutical composition or therapeutical composition of thepresent invention may comprise additives such as antiseptics, hydratesor emulsion accelerators, salts for the regulation of osmotic pressureand/or auxiliary such as buffers, and other therapeutically usefulsubstances. It can be prepared as formulations according to conventionalmethods.

The agonist ligands specific to G2A receptor as an active ingredient ofthe pharmaceutical composition or therapeutical composition of thepresent invention can be administered into mammals including humans viaparenteral or oral route in an amount of 0.01 to 100 mg/kg (body weight)once or several times a day. The extent of administered amount may varysuitably by the age, body weight, health condition, sex, the degree ofdisease, diet, administration time, excretion rate, administration routeand so on. The pharmaceutical composition or therapeutical compositionof the invention is not restricted to special dosage, administrationroute and administration method as far as it retains the inventiveeffects. Furthermore, the agonist ligands specific to G2A receptoraccording to the present invention may be co-administered together withantibiotics or therapeutic agents for inflammation that are generallyused for inflammation diseases when applied to inflammatory diseases,and co-administered together with various antibacterial agentscomprising the previous antibiotics when applied to treat a diseaseassociated with microbial infection.

The agonist ligands specific to G2A receptor according to the presentinvention can be effectively used for the treatment of a disease ordisorder associated with neutrophil accumulation due to suppression ofapoptosis and neutrophil hyperactivity and/or the excessive release ofIL-8, especially inflammatory diseases. The inflammatory diseases towhich the agonist ligands according the present invention can be appliedinclude all of the acute or chronic inflammatory diseases associatedwith suppression of neutrophil apoptosis and hyperactivity of neutrophiland/or the excessive release of IL-8, and complication thereof. The‘chronic inflammation’ refers to all diseases that induce tissue injuryor induce continuous inflammation due to excessive neutrophilaccumulation and hyperactivity and the excessive release of IL-8, andcomplication thereof. In particular, the inflammatory diseases to whichthe agonist ligands of the invention can be applied are not limited to,but include inflammatory bowel disease such as Crohn's disease andulcerative colitis, peritonitis, osteomyelitis, cellulitis, meningitis,cerebritis, pancreatitis, trauma-inducing shock, bronchial asthma,allergic rhinitis, cystic fibrosis, cerebral apoplexy, acute bronchitis,chronic bronchitis, acute bronchiolitis, chronic bronchiolitis,osteoarthritis, gout, spinal arthropathy, ankylosing spondylitis,Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis,juvenile arthropathy, juvenile ankylosing spondylitis, reactivearthropathy, infectious arthritis, post-infectious arthritis, gonococcalarthritis, tuberculous arthritis, viral arthritis, fungal arthritis,syphilitic arthritis, Lyme disease, arthritis associated with‘vasculitis syndrome’, polyarteritis nodosa, hypersensitivityvasculitis, Wegener's granulomatosis, polymyalgia rheumatica, giant cellarteritis, calcium crystal deposition arthropathy, pseudogout, non-jointrheumatism, bursitis, tenosynovitis, epicondylitis (tennis elbow),neuropathic joint disease(charcot joint), hemarthrosic, Henoch-Schonleinpurpura, hypertrophic osteoarthropathy, multicentricreticulohistiocytoma, scoliosis, hemochromoatosis, meniscocytosis, otherhemoglobinopathy, hyperlipoproteinemia, hypogammaglobulinaemia, familialmediterranean fever, Gerhardt Disease, systemic lupus erythematosus,relapsing fever, psoriasis, multiple sclerosis, sepsis (septicemia),septic shock, acute respiratory distress syndrome, multiple organdysfunction syndrome, chronic obstructive pulmonary disease, rheumaticarthritis, acute lung injury, bronchopulmonary dysplasia and so on.

In addition, it was reported that in patients with ischemia-reperfusioninjury, neutrophils was excessively accumulated. That is, almost all theorgans and tissues including heart, brain, kidney, liver, etc. aresubject to tissue damages owing to reperfusion when the disorder ofblood stream occurs. It has been known that the neutrophil has animportant role in this process (Jordan et al., Cardiovasc. Res., 43,860-78, 1999). Accordingly, the agonist ligands according to the presentinvention can be applied to treat ischemia-reperfusion injury includingischemic brain disease, ischemic heart disease, ischemic kidney disease,ischemic liver disease, ischemic bowel disease, organ injury due to thedisorder of blood stream in transplantation, etc. as well asinflammatory diseases.

Further, the present invention provides a therapeutic use of the agonistligands specific to G2A receptor. Specifically, the present inventionprovides a use of the agonist ligands specific to G2A receptor for themanufacture of a pharmaceutical composition for recovering thesuppressed apoptosis of neutrophils, inhibiting release of IL-8, orincreasing the bactericidal activity of neutrophils, in cells, tissuesor a body. The pharmaceutical composition may further comprisepharmaceutically acceptable carriers in addition to LPC (formula I), SPC(formula II) or derivatives thereof, preferably ether derivatives of LPC(formula III). The examples of the pharmaceutical acceptable carriersare as listed above. The pharmaceutical composition according to thepresent invention can be administered orally or parenterally, and theexamples of the oral or parenteral administration are as mentionedabove. Also, the present invention provides a use of the agonist ligandsspecific to G2A receptor for the manufacture of an agent for treating adisease or disorder associated with suppression of neutrophil apoptosisand hyperactivity of neutrophil and/or excessive release of IL-8. Theexamples of diseases or disorders to which the agonist ligands accordingto present invention can be applied are as listed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibitory effects of 1-stearoyl LPC againstsuppression of neutrophil apoptosis that is induced by an exogenousinflammation mediator (LPS; A) or endogenous inflammation mediators(GM-CSF, G-CSF and IFN-γ; B).

FIG. 2 shows the inhibitory effects of 1-stearoyl LPC against theexcessive release of IL-8 that is induced by LPS in neutrophils (A) andmonocytes (B).

FIG. 3 shows the RT-PCR results obtained using RNA isolated fromneutrophils as a template and primers specific to G2A- or GPR4receptors.

RT(+): RT-PCR is performed with reverse transcriptase.

RT(−): RT-PCR is performed without reverse transcriptase (Control group)

FIG. 4 shows the inhibitory effects of SPC against suppression ofneutrophil apoptosis that is induced by LPS.

FIG. 5 shows the effects of 1-stearoyl LPC in E. coli-induced septicemiamodel.

A: The survival rate of mice as time lapses after the administration ofLPC

B: The number of intraperitoneal E. coli cells in mice 24 hours afterLPC is administered

FIG. 6 shows the effects of 1-steroyl LPC in CLP-induced septicemiamodel.

A: The numbers of intraperitoneal E. coli cells in mice when LPC isadministered 2 hours and 14 hours after the CLP surgery, respectively

B: The survival rates of mice as time lapse when LPC is administeredfour times at intervals of 12 hours, 2 hours after the CLP surgery

C: The survival rates of mice as time lapse when LPC is administeredfour times at intervals of 12 hours, 10 hours after the CLP surgery.

FIG. 7 shows the effects of 1-myristoyl LPC (14:0 LPC) and 1-oleoyl LPC(18:1 LPC) in CLP-induced septicemia model.

FIG. 8 shows the effects of SPC in CLP-induced septicemia model.

FIG. 9 shows the effects of 1-stearoyl LPC against acute respiratorydistress syndrome in local LPS-induced acute lung injury model (A),systemic LPS-induced acute lung injury model (B) and acute lung injurymodel by CLP-induced sepsis (C).

FIG. 10 shows the effects of 1-stearoyl LPC against multiple organdysfunction syndrome in CLP-induced sepsis model.

FIG. 11 shows the effects of 1-stearoyl LPC regarding bactericidalactivity of neutrophils in bacteria-induced sepsis model.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described by virtue of the followingexamples in more detail.

However, the examples shown below are provided solely to illustrate theinvention; the scope of the invention should not be construed to belimited thereto. In the following examples, percentages with regard tosolid/solid mixture, liquid/liquid, and liquid/solid are based onweight/weight, volume/volume, and weight/volume, respectively, and allreactions were carried out at room temperature unless indicatedotherwise.

EXAMPLE 1

Inhibitory Effects of LPC against Anti-apoptosis of Neutrophils byInflammation Mediators

<1-1> Isolation of Neutrophils

Neutrophils were isolated from healthy adults using discontinuouspercoll gradient according to the method of Szucs, S. et al. (J.Immunol. Methods, 167: 245-251, 1994). First, plasmas were separatedfrom heparinized whole blood, and red blood cells were depleted bydextran sedimentation. Thereafter, leukocytes were layered over apercoll gradient with densities of 1.077 and 1.094 (Pharmacia, Sweden).Separated neutrophil layers were recovered from the percoll gradientinterface, and then washed with HBSS (Hank's balanced salt solution,Sigma Chemical Co., USA). The remaining pellet was resuspended in RPMI1640 medium supplemented with 10% FBS (fetal bovine serum) and 2 mMgentamicin. As a result of the examination of the cytospin stained withWright-Giemsa (Sigma Chemical Co., USA), it was observed that theresuspension solution contained 95% or more neutrophils. Also, as aresult of examination using trypan blue dye exclusion method, 98% ormore neutrophils were observed.

<1-2> Inhibitory Effects of LPC Against Anti-Apoptosis of Neutrophils byExogenous Inflammation Mediator

1×10⁵ neutrophil cells isolated in above Example <1-1> were cultured ina 96-well plate to which RPMI1640 medium containing 10% FBS was added.Thereafter, they were treated with 1-stearoyl lysophosphatidylcoline(Sigma Chemical Co., USA) in concentrations of 15 μM and 30 μM,respectively. After 1 hour, they were treated with 1 μg/ml LPS (SigmaChemical Co.). After 24 hours, the medium was removed from the plats,and the remaining cells were washed twice with PBS (phosphate-bufferedsaline) buffer at 4° C. The cells adhered to the bottom of the platewere all scraped and put into a 1.5-ml eppendorf tube. Then, they werecentrifuged at 2500 rpm for 5 min, and the supernatant was removed.Again, 0.1 ml of PBS buffer at 4° C. was added to the pellet, which wasthen well mixed using pipetting. To 25 μl of the cell suspension wasadded 2 μl of AcOr/EtBr dye solution in which 100 μg/ml of acridineorange (AcOr) and 100 μg/ml of ethidium bromide (EtBr) were mixed. Then,the percentage of apoptotic cells/total cells was calculated withobserving the neutrophil cells under a fluorescence microscope. As aresult, as shown in FIG. 1A, the percentage of neutrophil apoptosis thathad been decreased by LPS was increased by 1-stearoyl LPC in aconcentration-dependent manner. Accordingly, it can be seen that LPCeffectively blocks the effect of exogenous inflammation mediator thatinhibits neutrophil apoptosis.

<1-3> Inhibitory Effects of LPC Against Anti-Apoptosis of Neutrophils byEndogenous Inflammation Mediators

The experiments were carried out according to the same methods as usedin Example <1-2> except that the neutrophils isolated in Example <1-1>were treated with 100 ng/ml GM-CSF (Sigma Chemical Co.), 100 ng/ml G-CSF(Sigma Chemical Co.) and 100 ng/ml IFN-γ (Sigma Chemical Co.),respectively, instead of LPS. As a result, as shown in FIG. 1B, thepercentage of neutrophil apoptosis that had been decreased by endogenousinflammation mediators was increased by 1-stearoyl LPC in aconcentration-dependent manner. Accordingly, it can be seen that LPCeffectively blocks the inhibitory effects of neutrophil apoptosis byendogenous inflammation mediators as well as exogenous inflammationmediators.

EXAMPLE 2

Inhibitory Effects of LPC against Release of IL-8, which is anInflammation-Inducing Cytokine

Regarding release of IL-8, an important inflammation-inducing cytokinein neutrophils and blood monocytes, which is seriously considered inonset mechanism of inflammatory diseases, the effects of LPC wereinvestigated.

<2-1> Isolation of Neutrophils

Neutrophils were isolated according to the method of Szucs, S. et al.(J. Immunol. Methods, 167: 245-251, 1994) as described in above Example<1-1>.

<2-2> Isolation of Monocytes

Peripheral bloods were collected from a healthy person, put into aheparinized tube and then well mixed with 1× PBS buffer. Then, they werelayered in Ficoll-Hypaque solution (density 1.077, Sigma Chemical Co.,USA) and then centrifuged at 2500 rpm at 4° C. for 25 min. After themonocyte layer between Ficoll layer and plasma layer had been cautiouslyrecovered, 1×PBS buffer was added thereto and mixed well. It was thencentrifuged again at 2500 rpm at 4° C. for 25 min. Such centrifugationprocedures were repeated twice until red blood cells were depleted.Thereafter, the pellet was cultured in FBS at 37° C. for 15 min andcentrifuged at 2500 rpm for 10 min. Then, 1×PBS buffer was added againto the pellet. The isolated cells were layered in percoll solution(density 1.066) and then centrifuged at 2500 rpm at 4° C. for 30 min.After only monocyte layer had been recovered from the isolated layers,1×PBS buffer was added thereto. They were centrifuged at 2500 rpm for 15min. After the centrifugation had been repeated twice or three times,the isolated monocytes were inoculated into RPMI1640 medium containing10% FBS and cultured in an incubator of 5% CO₂ at 37° C.

<2-3> Measurement of IL-8

The neutrophils and monocytes isolated in Examples <2-1> and <2-2> abovewere cultured in a 96-well plate to which RPMI1640 medium containing 10%FBS was added, at a concentration of 1×10⁵ cells/well/0.2 ml,respectively. They were treated with 1-stearoyl LPC at concentrations of15 μM and 30 μM, respectively. After 1 hour, they were treated with 1μg/ml LPS. After 3 hours, supernatants were collected and put into aneppendorf tube. Then, the level of IL-8 was measured using ELISA kit(Biosource, USA). As a result, as shown in FIG. 2, the level of IL-8release that had been increased by LPS in neutrophils (A; human PMNs)and monocytes (B; human PBMC) was inhibited by LPC in aconcentration-dependent manner.

EXAMPLE 3

Search for LPC-Specific Receptors that Exist in Neutrophils

Until now, G2A and GPR4 have been known as receptors to which LPCspecifically binds (Zhu, K. et al., J. Biol. Chem., 276(44):41325-41335, 2001; Kabarowski, J. H. et al., Science, 293(5530):702-705, 2001). Especially, the fact that G2A exists in monocytes as areceptor of LPC was reported by Rikitake et al. (Rikitake et al.,Arterioscler Thromb Vasc Bio.l, 22: 2049-53, 2002). However, inneutrophils, nothing has been known. Accordingly, the present inventorsinvestigated whether LPC-specific receptors are in neutrophils.

First, neutrophils were isolated from human bloods according to the samemethods in above Example <1-1>. RNA was then isolated from the isolatedneutrophils using TRIZOL reagent (GibcoBRL, USA). RT-PCR was carried outusing primers specific to G2A (SEQ ID NO: 1 and SEQ ID NO:2) and primersspecific to GPR4 (SEQ ID NO: 3 and SEQ ID NO: 4), respectively. Thesizes of G2A and GRP4 to be amplified by each primer were 409 bp and 247bp, respectively. The PCR condition was as follows: 40 cycles of 94°C./2 min, 60° C./1.5 min and 72° C./2 min. As a control, RT-PCR wascarried out without reverse transcriptase (RT). As a result, as shown inFIG. 3, the expression level of G2A was remarkably increased as comparedwith the control group. On the other hand, the expression level of GPR4showed no difference from that of the control group. The expression ofeach receptor observed in the control group is regarded as being due tothe fact that DNA was completely not removed during the RNA isolationprocess. From these experiment results, it is considered that LPC doeshave inhibitory function of anti-apoptosis and inhibit release of IL-8by acting upon G2A receptor.

EXAMPLE 4

Inhibitory Effects of SPC against Anti-apoptosis of Neutrophils byInflammation Mediators

In order to examine whether SPC, known as another agonist ligand for G2Areceptor, does have the same functions as LPC, experiments were carriedout according to the same methods as used in above Example <1-2> exceptthat instead of LPC, SPC (Sigma-Aldrich Co.) was used. The results wereshown in FIG. 4. As shown in FIG. 4, the percentage of neutrophilapoptosis that had been decreased by LPS was increased by SPC in aconcentration-dependent manner. Accordingly, it can be seen that SPC,another ligand specific to G2A receptor, has the same functions as LPC.

REFERENCE EXAMPLE 1

Preparation of Septicemia Model

<1-1> Directly Induced Sepsis Model: Bacteria-induced Sepsis Model &LPS-induced Sepsis Model

a) Bacteria-Induced Sepsis Model

ICR mice (25-30 g in body weight; MJ Ltd.) were intraperitoneallyinjected with alive 10⁸ cells/ml E. coli (DH5a) that were suspended in0.5 ml of PBS (phosphate-buffered saline) to cause peritonitis, therebyinducing sepsis.

b) LPS-Induced Sepsis Model

ICR mice (25-30 g in body weight; MJ Ltd.) were intraperitoneallyinjected with LPS (induced from E. coli 055:B5) in an amount of 1 mg/kgthereby inducing Sepsis.

<1-2> Indirectly Induced Sepsis Model: CLP-Induced Sepsis Model

After ICR mice (25-30 g in body weight; MJ Ltd.) had been anesthetizedwith pentobarbital, right abdominal sites of the mice were dissected ina length of 1 cm to expose their cecum, and sites below the ileocecalvalve were ligated. And then, after 4˜6 punctures had been created onthe cecum with 21-gauge needle, the abdomen was sutured again to causeperitonitis, thereby inducing Sepsis.

REFERENCE EXAMPLE 2

Determination of the Activity of Myeloperoxidase in Pulmonary Tissues

The activity of myeloperoxidase (MPO), an indicator of neutrophils, inpulmonary tissues was determined according to the methods of Goldblum etal (J. Appl. physiol. 59: 1978, 1985) and Parey et al (J. Immunol.160:1007, 1998). First, mice were sacrificed and pulmonary tissues wereobtained therefrom. The tissues had been homogenized in potassiumphosphate buffer, and centrifuged to obtain pellets. These pellets weresonicated in potassium phosphate buffer containing 0.5%hexadecyltrimethylammonium bromide (HTAB), and incubated at 60° C. for120 min. After centrifugation, supernatants were obtained. 0.02 ml ofsupernatants was mixed with potassium phosphate buffer solution (0.18ml) containing o-dianisidine dihydrochloride of 0.167 mg/ml and 0.0005%hydrogen peroxide. Accordingly, the absorbance of the mixture wasdetermined at a wavelength of 460 nm.

REFERENCE EXAMPLE 3

Determination of the Number of Intraperitoneal E. coli

After the peritoneal cavity of mice had been exposed, it was washed withsterilized saline (2 ml). The resultant solution was diluted with HBSS(Sigma Chemical Co., USA) in a ratio of 1/1000, and then 10 μl of eachdiluted solution was spread on Trypticase Soy agar (BBL, BectonDickinson Co., USA) plates. After overnight incubation at 37° C., colonyforming unit (CFU) was measured.

EXAMPLE 5

Therapeutic Effects of 1-Stearoyl LPC in Bacteria-Induced Sepsis Model

14 ICR mice of bacteria-induced septicemia model according to ReferenceExample <1-1> a) were divided into two groups of 7 mice. 7 mice wereadministered with 1-stearoyl LPC (Sigma Chemical Co., USA) dissolved in1% BSA (bovine serum albumin) solution containing no fatty acid in anamount of 10 mg/kg by subcutaneous injection, 2 and 14 hours after E.coli was intraperitoneally injected, respectively (experimental group).The remaining 7 mice were administered with 1% BSA solution containingno fatty acid of the same amount as above, instead of LPC, in the samemanner (control group). Accordingly, the survival rates of the mice inthe experimental- and control groups over the lapse of time wereexamined. Also, the number of intraperitoneal E. coli cells 24 hoursafter the intraperitoneal injection was measured in accordance with themethod described in Reference Example 3. The average values werecalculated and exhibited in FIG. 5. As shown in FIGS. 5A and 5B, in themice of experimental group where LPC was administered, death rate due tosepticemia was significantly suppressed (P<0.05) and the number ofintraperitoneal E. coli cells was also remarkably decreased (P<0.01).

EXAMPLE 6

Therapeutic Effects of 1-Stearoyl LPC in CLP-Induced Sepsis Model

<6-1> In the Case where LPC is Administered 2 and 10 Hours after CLPSurgery, Respectively

14 ICR mice of CLP-induced septicemia model according to ReferenceExample <1-2> were divided into two groups of 7 mice. 7 mice wereadministered with 1-stearoyl LPC (Sigma Chemical Co., USA) dissolved in1% BSA solution containing no fatty acid in an amount of 10 mg/kg bysubcutaneous injection 2 and 14 hours after the CLP surgery,respectively (experimental group). The remaining 7 mice wereadministered with 1% BSA solution containing no fatty acid of the sameamount as above in the same manner (control group). The numbers ofintraperitoneal E. coli cells in the ICR mice of experimental andcontrol groups were measured in accordance with Reference Example 3, 24hours after the CLP surgery. The average values were calculated andexhibited in FIG. 6A. As shown in FIG. 6A, it could be seen that in themice of experimental group where LPC was administered, the number ofintraperitoneal E. coli cells was remarkably decreased (P<0.01).

<6-2> In the Case where LPC is Administered Four Times at Intervals of12 Hours. 2 Hours after CLP Surgery

40 ICR mice of CLP-induced Sepsis model according to Reference Example<1-2> were divided into four groups of 10 mice. Each 10 ICR mice wereintraperitoneally administered with 1-stearoyl LPC (Sigma Chemical Co.,USA) dissolved in 1% BSA solution containing no fatty acid in amounts of5, 10, and 20 mg/kg, respectively, four times at intervals of 12 hours,2 hours after the CLP surgery (experimental group-1, -2 and -3). Theremaining 10 ICR mice were administered only with 1% BSA solutioncontaining no fatty acid (control group). And then, the survival ratesof mice in experimental- and control groups were investigated over thelapse of time. The results are shown in FIG. 6B. As shown in FIG. 6B,the survival rate of the mice in experimental groups where LPC wasadministered was much higher than that of the mice in control group.

<6-3> In the Case where LPC is Administered Four Times at Intervals of12 Hours. 10 Hours after CLP Surgery

In order to investigate whether or not LPC has a therapeutic effect evenon cases where septicemia is already developed, 20 ICR mice ofCLP-induced Sepsis model according to Reference Example <1-2> weredivided into two groups. 10 mice for the experimental group wereintraperitoneally administered with 1-stearoyl LPC (Sigma Chemical Co.,USA) dissolved in 1% BSA solution containing no fatty acid in an amountof 10 mg/kg, four times at intervals of 12 hours, 10 hours after the CLPsurgery. The remaining 10 mice for the control group were administeredonly with 1% BSA solution containing no fatty acid. Thereafter, thesurvival rates of ICR mice in experimental- and control groups wereinvestigated over the lapse of time. The results are shown in FIG. 6C.As shown in FIG. 6C, even when septicemia was further developed, LPCshowed the excellent therapeutic effect.

EXAMPLE 7

Therapeutic Effects of 1-Oleoyl-LPC and 1-Myristoyl-LPC in CLP-InducedSepsis Model

In order to investigate whether LPCs having other substituents (R₁) but1-stearoyl have an effect on septicemia, LPCs having 1-oleoyl or1-myristoyl were intraperitoneally administered in an amount of 10 mg/kgaccording to the same methods as used in Example <6-2>. As a result, asshown in FIG. 7, both 1-oleoyl LPC and 1-myristoyl LPC showed theexcellent therapeutic effect against Sepsis.

COMPARATIVE EXAMPLE

In Vitro Antibacterial Activity of LPC

1-stearoyl LPC (Sigma Chemical Co., USA) was added to LB media atconcentrations of 12, 60 and 300 mM, respectively, which were pouredonto 60-mm petridishes and hardened (experimental group-1, -2 and -3).Four petridishes per group were used. For a control group, only LB mediawas poured onto four 60-mm petridishes and hardened. The peritonealcavity of mice according to Example <6-1> was washed with sterilizedsaline. The resultant solution was diluted with HBSS in a ratio of1/100, and then 10 μl of the diluted solution was spread on the mediafor each group. Subsequently, after they had been incubated in abacteria incubator maintained at 37° C. for 12 hours, colony formingunit (CFU) was visibly measured. The results were summarized in Table Ibelow.

TABLE 1 In Vitro Antibacterial Activity of LPC Control ExperimentalExperimental Experimental Group Group-1 Group-2 Group-3 LPC conc. 0 μM12 μM 60 μM 300 μM CFU 498 ± 189 661 ± 285 639 ± 304 611 ± 281

As shown in Table I above, there appeared no in vitro directantibacterial effects of LPC against E. coli.

EXAMPLE 8

Effects of SPC in CLP-Induced Sepsis Model

CLP-induced Sepsis model mice were subcutaneously injected with SPC,instead of 1-stearoyl LPC, in dosages of 3 mg/kg and 30 mg/kg four timesat intervals of 12 hours, 2 hours after the CLP surgery, respectively.The survival rates of mice were investigated. As a result, as shown inFIG. 8, it could be seen that like LPC, SPC effectively inhibited deathrate due to CLP Sepsis at a dosage of 30 mg/kg.

EXAMPLE 9

Therapeutic Effects of LPC Against Acute Respiratory Distress Syndrome

<9-1> LPS-Induced Acute Lung Injury (ALI) Model

Mice were intratracheally administered with LPS to cause direct acutelung injury, thereby inducing acute respiratory distress syndrome.

ICR mice (25-30 g in body weight; MJ Ltd.) were divided into five groupsof 7 mice (Sham group, experimental groups-1, -2, -3 and a controlgroup). Mice of each group were anesthetized with pentobarbital, theirskins were dissected in a length of 1 cm to expose their bronchia. Themice in Sham group were intratracheally administered with PBS (50 μl)solution and then sutured, while the ICR mice in experimental- andcontrol groups were directly intratracheally administered with LPS (6μg/50 μl PBS buffer) and then sutured. Thereafter, the ICR mice in theexperimental groups-1, -2 and -3 were subcutaneously administered with1-stearoyl LPC (Sigma Chemical Co., USA) dissolved in 1% BSA solutioncontaining no fatty acid in amounts of 5, 10, and 20 mg/kg,respectively, 2 and 14 hours after LPS had been administered. On theother hand, the ICR mice in a control group were administered with 1%BSA solution containing no fatty acid of the same amount, instead ofLPC, in the same manner. At 24 hours after LPS had been administered,the activity of MPO, an indicator of neutrophils, in the pulmonarytissues of the ICR mice of each group was determined as described inReference Example 2, and the average values are exhibited in FIG. 9A. Asshown in FIG. 9A, when LPS was intratracheally administered, theactivity of MPO was significantly increased (p<0.001). In the ICR miceof experimental groups, the activity of MPO was suppressed in aconcentration-dependent manner as compared with the mice of controlgroup. When LPC was administered into ICR mice in an amount of 20 mg/kg(experimental group-3), the most excellent effects were achieved(p<0.01).

<9-2> Systemic LPC-Induced Acute Lung Injury Model

Indirect acute lung injury was caused by systemic LPS administration,whereby acute respiratory distress syndrome was induced.

ICR mice in LPS-induced septicemia model according to Reference Example<1-1> b) were divided into five groups of 7 mice (Sham group, anexperimental group-1, an experimental group-2, an experimental group-3and a control group). The mice in Sham group were intraperitoneallyadministered with PBS buffer (0.5 ml/100 g weight), instead of LPS. Themice in the experimental groups-1, -2 and -3 were subcutaneouslyadministered with 1-stearoyl LPC (Sigma Chemical Co., USA) dissolved in1% BSA solution containing no fatty acid in amounts of 5, 10, and 20mg/kg, respectively 2 and 14 hours after LPS had been administered. Onthe other hands, the mice in a control group were administered with 1%BSA solution containing no fatty acid of the same amount, instead ofLPC, in the same manner. At 24 hours after LPS had been administered,the activity of MPO, an indicator of neutrophils, in the pulmonarytissues of the mice of each group, was determined as described inReference Example 2. The average values are exhibited in FIG. 9B. Asshown in FIG. 9B, when LPS was intraperitoneally administered, theactivity of MPO was significantly increased (p<0.01). In the mice of theexperimental groups, the activity of MPO was suppressed in aconcentration-dependent manner as compared with the mice of a controlgroup. When LPC was administered into ICR mice in an amount of 20 mg/kg(experimental group-3), the most excellent effects were achieved(p<0.01).

<9-3> Acute Lung Injury Model by CLP-Induced Sepsis

The CLP-induced Sepsis model was used to cause indirect acute lunginjury, thereby inducing acute respiratory distress syndrome.

20 ICR mice of CLP-induced Sepsis model according to Reference Example<1-2> were divided into two groups. 10 mice for the experimental groupwere administered with1-stearoyl LPC (Sigma Chemical Co., USA) dissolvedin 1% BSA solution containing no fatty acid in an amount of 10 mg/kg bysubcutaneous injection 2 and 14 hours after the CLP surgery,respectively. The remaining 10 mice for the control group wereadministered only with 1% BSA solution containing no fatty acid. In themeantime, 10 ICR mice (25-30 g in body weight; MJ Ltd.) wereanesthetized with pentobarbital, of which right abdominal sites weredissected in a length of 1 cm to expose their cecum, and then suturedagain (Sham group). Right after CLP surgery and 4, 8 and 16 hours later,the activity of MPO, an indicator of neutrophils, in the pulmonarytissues of the mice of each group was determined as described inReference Example 2 and exhibited in FIG. 9C. As shown in FIG. 9C, theactivity of MPO was remarkably increased since 4 hours after the CLPsurgery and maintained till 16 hours. In the ICR mice of experimentalgroup, the activity of MPO, which had been increased by CLP surgery, wasremarkably suppressed as compared with the control group.

EXAMPLE 10

Therapeutic Effects of LPC Against Multiple Organ Dysfunction Syndrome

Septicemia causes multiple organ dysfunction syndrome as itscomplication and typically, lung, liver, kidney, etc. are injured. Theeffect of LPC against liver injury as one of the multiple organdysfuction syndromes was investigated. 20 ICR mice of CLP-inducedsepticemia model according to Reference Example <1-2> were divided intotwo groups. 10 mice for the experimental group were administered with1-stearoyl LPC (Sigma Chemical Co., USA) dissolved in 1% BSA solutioncontaining no fatty acid in an amount of 10 mg/kg by subcutaneousinjection 2 and 14 hours after the CLP surgery, respectively. Theremaining 10 mice for the control group were administered only with 1%BSA solution containing no fatty acid. In the meantime, 10 ICR mice(25-30 g in body weight; MJ Ltd.) were anesthetized with pentobarbital,of which right abdominal sites were dissected in a length of 1 cm toexpose their cecum, and then sutured again (Sham group). At 16 hoursafter the CLP surgery, blood was collected from mice in each group. Thelevel of alanine aminotransferase (ALT), an indicator of hepatotoxicity,was determined according to the Reitman-Frankel method (Witter & Grubbs,Clin Chim Acta, 13:524-7, 1966). As a result, as shown in FIG. 10, inthe ICR mice of experimental group, ALT level, which had been increasedby CLP surgery, was remarkably suppressed as compared with the controlgroup.

EXAMPLE 11

Effects of LPC on Bactericidal Activity of Neutrophils inBacteria-Induced Sepsis Model

In order to investigate mechanism on how the number of intraperitonealE. coli cells in mice was remarkably decreased due to the administrationof LPC in Example 5 above, the inventors conducted the followingexperiments: Blood was collected from bacteria-induced septicemia modelmice prepared in Reference Example <1-1> a), and neutrophils wereisolated therefrom. The isolated neutrophils were adhered to the coverslip coated with poly-L-Lysine (0.01%) in a cell incubator maintained at37° C., at a cell concentration of 10⁶ cells/ml for 1 hour. Thereafter,E. coli was added thereto in a concentration of 10⁶ cells/ml andcultured for 1 hour. E. coli cells that were not ingested by neutrophilswere removed by washing the cover slip with PBS buffer. The cover slipwas then treated with 30 μp M 1-stearoyl LPC, and further cultured foranother 1 hour. In this stage, the cover slip of the control group wastreated with PBS buffer, instead of LPC. Thereafter, after the exchangeof a new RPMI-1640 medium, they were further cultured for 1 hour to givekilling time. Then, the neutrophils were broken using Triton X-100(0.05%), and accordingly E. coli cells were recovered. The recovered E.coli cells were spread onto a plate and cultured in an incubatormaintained at 37° C. The number of E. coli cells that had grown on themedium was counted and subjected to statistical analysis. As a result,as shown in FIG. 11, in the experimental group that was treated withLPC, the number of alive E. coli was significantly decreased. Thisindicates that LPC increases the bactericidal ability of neutrophils bydirectly stimulating the neutrophils. Accordingly, it can be seen thatLPC blocks the suppressed neutrophil apoptosis, inhibits release of IL-8in neutrophils/monocytes and also increases the bactericidal activity ofneutrophils.

INDUSTRIAL APPLICABILITY

As described in the above, it was identified in the present inventionthat LPC, SPC and derivatives thereof, agonist ligands specific to G2Areceptor, can effectively inhibit the anti-apoptosis of neutrophils andthe excessive release of IL-8. In addition, it was revealed that theagonist ligands according to the present invention can effectivelyeliminate pathogens in microbial infection by increasing thebactericidal activity of neutrophils. Accordingly, the agonist ligandsspecific to G2A receptor according to the present invention andpharmaceutical- or therapeutical composition comprising the said ligandscan be used very effectively in treatment of a disease or disorderassociated with neutrophil accumulation and/or excessive release ofIL-8, especially inflammatory diseases including sepsis, or a diseaseassociated with microbial infection.

1. A method for treating sepsis or septic shock comprising administering to a subject in need thereof an effective amount of a compound selected from the group consisting of 18:0 LPC (1-stearoyl lysophosphatidylcholine), 18:1 LPC (1-oleoyl lysophosphatidylcholine), 14:0 LPC (1-myristoyl lysophospatidylcholine), 16:0 LPC (1-palmitoyl lysophosphatidylcholine) and SPC (sphingosylphosphorylcholine).
 2. The method of claim 1, wherein the effective amount of the compound is from 0.01 to 100 mg per kg of body weight of the subject in need thereof. 